1. Field of the Invention
A gold nanoparticle-based colorimetric assay for hepatitis C virus RNA that detects unamplified HCV RNA in clinical specimens using unmodified gold nanoparticles (“AuNPs”).
2. Description of the Related Art
Gold nanoparticles or AuNPs exhibit a unique phenomenon known as Surface Plasmon Resonance, which is responsible for their intense red color in suspension. This color changes to blue upon aggregation of AuNPs.
According to the World Health Organization, there are around 200 million people worldwide infected with hepatitis C virus (“HCV”), with three to four million newly infected patients annually [1]. HCV is a small enveloped single-stranded RNA virus that belongs to the Flaviridae family, Hepacivirus genus, which comprises a group of highly variable strains or isolates. HCV is a blood borne virus and infection has different clinical outcomes ranging from acute resolving hepatitis to chronic liver diseases, including liver fibrosis, cirrhosis and hepatocellular carcinoma [2; 3; 4]. The acute viral infection resolves in 15% of infected patients but progresses in 85% of patients to chronic infection [5].
Different HCV isolates frequently exhibit genetic differences and HCV isolates have been classified into six different genotypes, which are further grouped into subtypes identified by letter designations, e.g. HCV Genotype 1a. Different HCV genotypes predominate in particular parts of the world. For example, in North America genotype 1a predominates, followed by genotypes 2a, 2b, 2c, and 3a. Genotypes 4 and 5 are found almost exclusively in Africa. The genotype of HCV is clinically significant in determining therapeutic response, such as responses to interferon-based therapy. Prior infection with one HCV genotype does not necessarily confer protection against another. Genotypes have up to 67% percent nucleotide similarity to one another and subtypes have nucleotide similarity up to 78%. More than 80 different subtypes have been identified. HCV isolates that have sequence similarity between 91-99% are termed quasispecies.
Currently HCV is detected using immunoassays, such as antibody-based immunoassays and confirmed by molecular assays, such as RT-PCR. For example, immunoassays such as enzyme linked immunoassays (EIAs) and recombinant immune blot assays (RIBA) are used for detection of anti-HCV antibodies [6] and conventional RT-PCR and transcription-mediated amplification (TMA) are used for qualitative detection of HCV RNA while quantitative detection is achieved using real-time RT-PCR, and/or branched DNA-based assays [7].
Despite the high sensitivity and specificity of these methods, they are time-consuming, labor intensive, expensive, and require specialized equipment and thus are not suitable for use in many developing countries or for use in the field. Therefore, there is a great need to develop a low-tech assay for the direct detection of unamplified HCV RNA with acceptable sensitivity and specificity, short turnaround time, and cost-effectiveness. Such an assay would be critical to characterize and control HCV in developing countries with limited resources and high infection rates, such as Egypt.
Nanoparticles have been recently proposed as promising tools to develop the next generation of diagnostic assays. Because of their unique properties and ability to interact with biomolecules on one-to-one basis, various nanoparticles show great promise to meet the rigorous demands of the clinical laboratory for sensitivity and cost-effectiveness, and can be used in the future in point-of-care diagnosis [8].
Gold nanoparticles (“AuNPs”) are spheres with a typical diameter of approximately 2-50 nm. They exhibit a unique phenomenon known as Surface Plasmon Resonance (SPR), which is responsible for their intense red color, and which changes to blue upon aggregation of AuNPs [9].
The addition of salt shields the surface charge on the AuNPs, which are typically negatively charged owing to adsorbed negatively charged citrate ions on their surfaces, leading to aggregation of AuNPs and a red-to-blue color shift [10]. SPR is also responsible for the large absorption and scattering cross-sections of AuNPs which are 4-5 orders of magnitude larger than those of conventional dyes [11]. These unique optical properties have allowed the use of AuNPs in simple and rapid colorimetric assays for clinical diagnosis offering higher sensitivity and specificity than current detection techniques [12, 13]. Suitable components and procedures for making gold nanoparticles are known in the art and are incorporated by reference to the articles cited herein.
Li et al. developed a colorimetric assay using unmodified citrate-coated AuNPs [10, 13]. This method is based on the property of single-stranded DNA (ssDNA) which adsorbs on citrate-coated AuNPs. This adsorption increases the negative charge on the AuNPs leading to increased repulsion between the particles, thus preventing aggregation. The adsorption of ssDNA on AuNPs occurs due to the fact that ssDNA can uncoil and expose its nitrogenous bases. The attractive electrostatic forces between the bases and the AuNPs allow adsorption of the ssDNA. On the other hand, double-stranded DNA (dsDNA) does not adsorb on AuNPs due to the repulsion between its negatively-charged phosphate backbone and the negatively-charged coating of citrate ions on the surfaces of the AuNPs. Therefore, when AuNPs are added to a saline solution containing the target DNA and its complementary unlabeled single-stranded polynucleotide, AuNPs aggregate (since the single-stranded polynucleotides are not free to stabilize the AuNPs) and the solution color changes to blue. However, in the absence of the target or the presence of a non-complementary target, the complementary single-stranded polynucleotides are free to stabilize the AuNPs thus preventing their aggregation and the solution color remains red. This method has been used to detect single nucleotide polymorphisms in PCR-amplified genomic DNA extracted from clinical samples [10]. Moreover, based on the same principle, AuNPs are capable of quenching fluorescent dyes and this property has been used for detection of synthetic HCV sequences with high sensitivity and selectivity [14, 15]. Shawky, et al., Clin. Biochem. 43:1163-1168 (2010), which is incorporated by reference, discloses direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles.